Mass Spectrometry Detection and Imaging of a Non‐Covalent Protein–Drug Complex in Tissue from Orally Dosed Rats

Abstract Here, we demonstrate detection by mass spectrometry of an intact protein–drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein–drug complex comprised fatty acid binding protein 1, FABP1, non‐covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non‐covalent protein–drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target‐drug characterization directly from the physiological environment.


Supporting Information
. Structure of bezafibrate.              Six male Han-Wistar rats (weight range 200-220g) were obtained from Charles River Laboratories (Margate, Kent, UK). Vendor-supplied health reports indicated that the rats were free of known viral, bacterial and parasitic pathogens. They were housed in groups of 3 in Tecniplast IVC cages (model number GR1800DD), in a 12 h light: dark cycle with room temperature maintained at 21°C +/-2°C and relative humidity maintained at 55% +/-10%, in a semi-barrier facility. The cages contained a 1-1.5 cm layer of 4 mm 3 Aspen chip bedding together with environmental enrichment (nestlet nesting material, Datesand) medium Aspen brick chew sticks (Datesand) and a cardboard play tunnel (Datesand). The rats had access to food (RM1 (E) IRR 0.25 pelleted diet, Special Diet Services, UK) and water ad libitum. Rats were allocated to cages on arrival and remained in the same social group throughout the study. Rats were test-naïve prior to the studies. Animal welfare was assessed throughout by daily monitoring of appearance, behaviour and cage environment. Rats were allowed to acclimatise to the animal unit for at least 1 week prior to in vivo work and were approximately 250g at experiment start.
Animal experiments and tissue dissection were performed by trained AstraZeneca staff. Rats were allocated to two groups: one group was euthanised 2 hours after dosing; the second group was euthanised 6 hours after dosing. In each group, two animals were dosed with the drug and one animal was vehicle-dosed (control). Bezafibrate was purchased from Sigma-Aldrich (Gillingham, UK) and a single oral dose of 150 mg/kg, (Vehicle 0.5% hydroxypropyl methycellulose (HPMC) and 0.1% Tween anaesthetised by administration of isofluorane, then euthanised by cutting the descending aorta and vena cava and death was confirmed by removal of the brain. Tissue samples were collected immediately and snap-frozen in isopentane over dry ice. Left lobes of livers were cut transversally, placed in ziplock bags to avoid deformations and stored at -80°C. Tissue sections were prepared 1-4 days before mass spectrometry analysis by cryosectioning (CM1810 Cryostat, Leica Microsystems, Wetzlar, Germany) at a thickness of 10 µM and thaw mounting onto glass slides. Sections were labelled, wrapped in aluminium foil and stored at -80°C until analysis. No washing procedures were performed to avoid any potential disruption of protein structure.
Liquid extraction surface analysis (LESA)  m, I.D. 75 m prior to modification) and cut to a final outer diameter of approx. 100 m. The sampling capillary was positioned directly in front of the mass spectrometer inlet to aspirate solvent with the inlet vacuum. The exit of the sampling capillary was not flame-pulled but had its coating removed.
Solvent was delivered at a rate of 1.9 L/min through the first capillary by a 10 mL gas-tight syringe (Hamilton, Reno, NV) and a liquid junction was formed between the two capillaries. The liquid junction covered a region of tissue approx. 200 m in diameter. High voltage was supplied from the mass spectrometer to the syringe needle and optimized for ion intensity and spray stability (0.8 kV for MSI).
The ion transfer tube was set to 250 °C.

Mass spectrometry
Positive ion mode full scan mass spectra were collected by use of an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with settings as follows: 100% AGC target, 60 RF%, 2 microscans, 500 ms injection time, 120,000 resolution, ions were detected with the orbitrap in the range of m/z 300-3000 using protein mode, 250°C ion transfer tube temperature, standard pressure. All mass spectra were analysed in FreeStyle v. 1.4 (Thermo Fisher Scientific) and exported as csv files for subsequent plotting in GraphPad Prism v. 6.1 and Origin 2016.
SIM-mode mass spectra were also acquired by use of the Orbitrap Eclipse mass spectrometer with settings as follows: AGC target 100%, RF 60%, microscans 2, resolution 120,000, SIM scan mode, scan low m/z 1831.5, scan high m/z 1839.5, protein mode, standard pressure, 250°C ion transfer tube temperature. Mass spectra presented comprised 50 summed scans. Mass spectra were analysed in Nano-DESI MSI was performed on the Orbitrap Eclipse mass spectrometer by multiplexed targeted proton transfer charge reduction (tPTCR) of intact proteins. S-lens RF was set to 40%, and source collision voltage was "off". Precursor ions for three proteins and the [FABP1-bezafibrate] complex (see Table S2, Supporting Information) were isolated sequentially for up to 250 ms using the quadrupole mass analyser and stored together in the ion routing multipole in N2 bath gas set to a pressure of 8 mTorr. All isolated ions were then passed to the high-pressure cell of the linear ion trap and underwent PTCR for 7 ms with approx.           Figure S4.     were between 3-5 ppm. * indicates monoisotopic peak, * indicates 37 Cl isotope peak. Figure S10. Replicate SIM-mode mass spectra obtained from three separate locations in liver sections from Animal 4 (2 hour timepoint) and Animal 6 (6 hour timepoint). m/z of most abundant isotope peak is given.